Rational design of enzymes with improved properties, such as enantioselectivity, usually focuses mutations within the substrate binding site. On the other hand, directed evolution of enzymes usually targets the entire protein and discovers beneficial mutations far from the substrate binding site. In this paper, we propose an explanation for this discrepancy and show that a combined approach - random mutagenesis within the substrate binding site - is better. To increase the enantioselectivity (E) of a Pseudomonas fluorescens esterase (PFE) toward methyl 3-bromo-2-methylpropionate, we focused mutagenesis into the substrate binding site at Trp28, Val121, Phe198, and Val225. Five of the catalytically active mutants (13%) showed better enantioselectivity than wild-type PFE. The increases in enantioselectivity were higher (up to 5-fold, reaching E = 61) than with mutants identified by random mutagenesis of the entire enzyme.
Bibliographical noteFunding Information:
We thank the Natural Sciences and Engineering Research Council of Canada for financial support and a fellowship to K.L.M. and the Swedish Foundation for International Cooperation in Research and Development for financial support. We thank Prof. Dr. Uwe T. Bornscheuer (Ernst-Moritz-Arndt-University, Greifswald, Germany) for the expression plasmid for PFE and Linda Fransson (Royal Institute of Technology, Stockholm, Sweden) for assistance in constructing the homology model.
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